It is often desirable to detect certain known or suspected target sequences within samples of DNA that may be derived from biological sources (either directly, or by indirect methods, e.g., reverse transcription of DNA), or which may be derived by artificial means, e.g., chemical synthesis or site-directed mutagenesis. Detection of such target sequences may have utility in determining the presence of infectious diseases such as HIV-I or Hepatitis B virus, as well as in the detection of whether individuals carry genes for genetic diseases such as sickle cell anemia or hemophilia. Such methods may also be useful in identifying whether target sequences are contained in populations of DNA produced by genetic manipulations in vitro.
Perhaps the most powerful methods for detecting such target DNA sequences take advantage of existing and emerging methods for amplifying DNA sequences that may be present in samples in only trace quantities. One particularly well-known amplification method is the polymerase chain reaction (PCR). Once amplified, target DNA sequences can be detected by a variety of methods for the detection of specific DNA sequences, e.g., by gel electrophoresis or by hybridization with labeled probes, or, if the amplified product is present in substantially large quantities in relationship to the DNA present in the original sample, by more simple methods that detect the relative presence of DNA, e.g., by staining with ethidium bromide, which becomes fluorescent upon intercalation between nucleotide bases in double-stranded DNA.
Unfortunately, available methods for detecting DNA amplification products have limitations that diminish the usefulness of such methods for identifying target sequences. For example, gel electrophoresis requires a considerable amount of sample handling, and is thus not suitable for the rapid and cost-effective screening of large numbers of samples. This handling can also lead to false positives if even extremely small amounts of DNA are carried over from sample to sample, as these may become subsequently amplified.
A sense of the importance of such detection methods and the limitations of those currently available can be readily appreciated in the case of methods used to detect the presence of specific HLA-class II molecules. These molecules are highly polymorphic antigens which play a key role in the control of the immune response. For example, the HLA-class II molecules DR and DQ are involved in causing tissue rejection after tissue transplantation, auto-immune diseases, and other immune-mediated disorders. There is therefore a clinical need to be able to detect the presence of these antigens in given individuals, in order to allow for tissue type matching in anticipation of organ transplantation, investigations into auto-immune and other HLA-related diseases, and studies designed to explore the evolution and descendance of these antigens.
Since the development of PCR (1,2), many amplification-dependant approaches have been applied to HLA typing. For example, restriction endonuclease digestion to produce PCR restriction fragment length polymorphism (PCR-RFLP) has been used (3,4), and an even more popular approach has been the hybridization of PCR amplified products with sequence-specific oligonucleotide probes (PCR-SSO) to distinguish between HLA alleles (5-7). Hybridization and detection methods for PCR-SSO typing include the use of non-radioactive labeled probes (8,9), microplate formats (10-12), reverse dot blot formats (13,14) and automated large scale HLA class 11 typing (15). A common drawback to these methods, however, is the relatively long assay times needed--generally one to two days--and their relatively high complexity and resulting high cost. In addition, the necessity for sample transfers and washing steps increases the chances that small amounts of amplified DNA might be carried over between samples, creating the risk of false positives.
Recently, a molecular typing method using sequence specific primer amplification (PCR-SSP) has been described (16-18). This PCR-SSP method is simple, useful and fast relative to PCR-SSO, since the detection step is much simpler. In PCR-SSP, sequence specific primers amplify only the complementary target allele, allowing genetic variability to be detected with a high degree of resolution. This method allows determination of HLA type simply by whether or not amplification products (collectively called an "amplicon") are present or absent following PCR.
In PCR-SSP, detection of the amplification products is usually done by agarose gel electrophoresis followed by ethidium bromide (EtBr) staining of the gel. Unfortunately, the electrophoresis process takes a long time and is not very suitable for large number of samples, which is a problem since each clinical sample requires testing for many potential alleles. Gel electrophoresis also is not easily adapted for automated HLA-DNA typing.
More recently, HLA-DNA PCR-SSP typing using Ethidium homodimer (EthD) staining without electrophoresis has been described (19-20). These methods still require the transfer of PCR products, and this handling increases the chance that traces of amplified DNA will be transferred from sample to sample, which can lead to contamination and false positives.
In an effort to eliminate the need for sample transfers, a homogeneous method for the detection of PCR amplified products using Ethidium bromide (EtBr) fluorescence detection has been developed (21,22). In this method, ethidium bromide is simply added to the amplification reaction mixture; since the amplification product should be present in a large amount relative to the DNA of the starting sample, ethidium bromide fluorescence in a sample where amplification occurred, i.e. where the target sequence was present, is greater than the fluorescence in a sample where the target sequence was lacking, and amplification did not occur. Unfortunately, the template DNA, partial primer dimer, and primer present in both positive and negative samples represents a substantial background, making the discrimination between positive and negative samples somewhat difficult and unreliable. The end result is that the method has relatively low sensitivity and reproducability. Prior to the present invention, no means for reducing this background effect was known.